TCR-seq

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Workflow

Calling Clonotypes

Calling method from contigs

1. Using Gene information

  • VDJC(TRA/TRB)
  • CDR3nt
  • CDR3aa
  • Combination

2. Using Chain information

  • TRA1+TRB1
  • TRA1 or TRB1
  • multi-chains

Basic Clonotype Analysis

Immune repertoire analysis

1. Diversity Index

  • Shannon Entropy
  • Simpson

2. Repertoire analysis.

  • TRAV/D/J/C distribution
  • TRBV/D/J/C distribution
  • CDR3nt length distribution
  • Clustering of clonotype repertoire

3. Expansion study

  • Clonotype expansion (hyper,high,medium,small,rare)
  • Top 100 expanded clonotypes with TRAV:TRBV info


Target Cell:Clonotype Analysis

Combination with scRNA-seq (if available)

1. Target cell selection

  • Cells with target features (e.g. cytotoxic cells, effector T cells, NKT, etc).
  • Cells associated with expanded clonotypes (hyper, high, medium, small, rare).
  • Cells with clonotypes displaying treatment-specific response.

2. Correlation of clonotype frequency with related data

  • Correlation of clonotype frequency with target gene expression profiles.
  • Correlation with clinical outcomes and phenotypic data. (if available)

3. Comparative Analysis

  • Comparison between different patient groups (e.g., responders vs non-responders to therapy, different disease stages) and associated clonotypes.
  • Clonotype repertoires to identify conserved features of the immune response.

4. Statistical and Computational Approaches

  • Application of statistical models and computational algorithms for the analysis.
  • Evaluation of the robustness and reproducibility of findings.



Example Plots

Clonotype Distribution



Clonotype Abundance


Sample A

Sample B


Cytotoxic Gene Expression Profiling across Clonotypes and Samples

Important Cytotoxic Gene Information

Gene Description
NKG7 Natural Killer Growth Factor 7, the function of cytotoxic lymphocytes, particularly Natural Killer (NK) cells, potentially through the regulation of apoptotic pathways.
KLRG1 Killer Cell Lectin-Like Receptor G1, is a transmembrane protein receptor primarily expressed on cytotoxic lymphocytes, regulating their activation and effector functions.
IFNG Interferon Gamma, is a cytokine crucially involved in the activation and effector functions of cytotoxic lymphocytes, including NK cells and cytotoxic T cells. IFNG enhances the cytotoxicity of these cells, promotes antigen presentation, and directs T helper cell differentiation towards a Th1 phenotype, crucial for effective immune responses.
GZMA Granzyme A, is a serine protease predominantly expressed in cytotoxic lymphocytes, facilitating target cell apoptosis. Upon recognition of target cells, GZMA is released and initiates caspase-independent cell death pathways, leading to the elimination of infected or transformed cells.
PRF1 Perforin-1, is a pore-forming protein primarily expressed in cytotoxic lymphocytes, essential for target cell lysis. PRF1 forms pores in target cell membranes, enabling the delivery of pro-apoptotic molecules and promoting target cell death, crucial for immune surveillance and defense.



Heatmap